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MicrobiologyOpen Jun 2024The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present...
The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.
Topics: ADAM10 Protein; NLR Family, Pyrin Domain-Containing 3 Protein; Humans; Receptors, G-Protein-Coupled; Hemolysin Proteins; Inflammasomes; Bacterial Toxins; THP-1 Cells; Receptors, Estrogen; Amyloid Precursor Protein Secretases; Staphylococcus aureus; Membrane Proteins; Caspase 1; Adenosine Triphosphate; Macrophages; Dipeptides; Hydroxamic Acids
PubMed: 38867416
DOI: 10.1002/mbo3.1423 -
Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli.Microbial Cell Factories Jun 2024The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several...
BACKGROUND
The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme.
RESULTS
Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source.
CONCLUSIONS
This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.
Topics: Escherichia coli; Acetyl Coenzyme A; Citrate (si)-Synthase; 3-Hydroxybutyric Acid; Metabolic Engineering; Escherichia coli Proteins; Ketone Oxidoreductases; Alcohol Oxidoreductases
PubMed: 38867236
DOI: 10.1186/s12934-024-02444-8 -
Nature Communications Jun 2024Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating...
Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating invasive tumor indications such as melanoma. However, insufficient dose deposition and immunosuppressive microenvironment (TME) of solid tumors limit its efficacy. Here we report a programmable sequential therapeutic strategy based on multifunctional fusogenic liposomes (Lip@AUR-ACP-aptPD-L1) to overcome the intrinsic radio-immunotherapeutic resistance of solid tumors. Specifically, fusogenic liposomes are loaded with gold-containing Auranofin (AUR) and inserted with multivariate-gated aptamer assemblies (ACP) and PD-L1 aptamers in the lipid membrane, potentiating melanoma-targeted AUR delivery while transferring ACP onto cell surface through selective membrane fusion. AUR amplifies IR-induced immunogenic death of melanoma cells to release antigens and damage-associated molecular patterns such as adenosine triphosphate (ATP) for triggering adaptive antitumor immunity. AUR-sensitized radiotherapy also upregulates matrix metalloproteinase-2 (MMP-2) expression that combined with released ATP to activate ACP through an "and" logic operation-like process (AND-gate), thus triggering the in-situ release of engineered cytosine-phosphate-guanine aptamer-based immunoadjuvants (eCpG) for stimulating dendritic cell-mediated T cell priming. Furthermore, AUR inhibits tumor-intrinsic vascular endothelial growth factor signaling to suppress infiltration of immunosuppressive cells for fostering an anti-tumorigenic TME. This study offers an approach for solid tumor treatment in the clinics.
Topics: Liposomes; Aptamers, Nucleotide; Animals; Mice; Cell Line, Tumor; Immunotherapy; Melanoma; Humans; Tumor Microenvironment; Matrix Metalloproteinase 2; Gold; Mice, Inbred C57BL; Female; B7-H1 Antigen; Adenosine Triphosphate
PubMed: 38866788
DOI: 10.1038/s41467-024-49482-9 -
Scientific Reports Jun 2024Red blood cells (RBCs) exhibit an interesting response to hydrodynamic flow, releasing adenosine triphosphate (ATP). Subsequently, these liberated ATP molecules initiate...
Red blood cells (RBCs) exhibit an interesting response to hydrodynamic flow, releasing adenosine triphosphate (ATP). Subsequently, these liberated ATP molecules initiate a crucial interaction with endothelial cells (ECs), thereby setting off a cascade involving the release of calcium ions (Ca ). Ca exerts control over a plethora of cellular functions, and acts as a mediator for dilation and contraction of blood vessel walls. This study focuses on the relationship between RBC dynamics and Ca dynamics, based on numerical simulations under Poiseuille flow within a linear two-dimensional channel. It is found that the concentration of ATP depends upon a variety of factors, including RBC density, channel width, and the vigor of the flow. The results of our investigation reveals several features. Firstly, the peak amplitude of Ca per EC escalates in direct proportion to the augmentation of RBC concentration. Secondly, increasing the flow strength induces a reduction in the time taken to reach the peak of Ca concentration, under the condition of a constant channel width. Additionally, when flow strength remains constant, an increase in channel width corresponds to an elevation in calcium peak amplitude, coupled with a decrease in peak time. This implies that Ca signals should transition from relatively unconstrained channels to more confined pathways within real vascular networks. This notion gains support from our examination of calcium propagation in a linear channel. In this scenario, the localized Ca release initiates a propagating wave that gradually encompasses the entire channel. Notably, our computed propagation speed agrees with observations.
Topics: Erythrocytes; Adenosine Triphosphate; Calcium; Endothelial Cells; Humans; Calcium Signaling
PubMed: 38866785
DOI: 10.1038/s41598-024-63306-2 -
Proceedings of the National Academy of... Jun 2024The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential...
The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP] in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]. Notably, diastolic [ATP] was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K (K) channels were active at physiological [ATP]. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]. Mode 1 [ATP] increases necessitated Ca entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca. By contrast, decreases in mitochondrial Ca accompanied Mode 2 [ATP] decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP] fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of β-adrenergic receptors decreased [ATP], underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle.
Topics: Myocytes, Cardiac; Adenosine Triphosphate; Excitation Contraction Coupling; Animals; Calcium; Heart Ventricles; Action Potentials; Sarcoplasmic Reticulum; Heart Rate; Humans; KATP Channels; Myocardial Contraction; Mice
PubMed: 38865270
DOI: 10.1073/pnas.2318535121 -
Immunity, Inflammation and Disease Jun 2024This study aimed to link intracellular adenosine triphosphate content in CD4 T lymphocytes (CD4 iATP) with sepsis patient mortality, seeking a new predictive biomarker... (Observational Study)
Observational Study
OBJECTIVE
This study aimed to link intracellular adenosine triphosphate content in CD4 T lymphocytes (CD4 iATP) with sepsis patient mortality, seeking a new predictive biomarker for outcomes and enhanced management.
METHODS
61 sepsis patients admitted to the Intensive Care Unit between October 2021 and November 2022 were enrolled. iATP levels were gauged using whole blood CD4 T cells stimulated with mitogen PHA-L. Based on CD4 iATP levels (<132.24 and ≥132.24 ng/mL), patients were categorized into two groups. The primary endpoint was all-cause mortality. To identify factors associated with mortality, both univariate and multivariate Cox proportional hazard analyses were conducted.
RESULTS
Of the patients, 40 had high CD4 iATP levels (≥132.24 ng/mL) and 21 had low levels (<132.24 ng/mL). In a 28-day follow-up, 21 (34.4%) patients perished. Adjusting for confounders like SOFA score, APACHE II score, lactic acid, and albumin, those with low CD4 iATP had three- to fivefold higher mortality risk compared to high CD4 iATP patients (61.9% vs. 20.0%; hazard ratio [95% confidence interval], Model 1: 4.515 [1.276-15.974], p = .019, Model 2: 3.512 [1.197-10.306], p = .022). CD4 iATP correlated positively with white blood cell and neutrophil counts but not with lymphocytes, CD3, and CD4 counts.
CONCLUSIONS
Low CD4 iATP levels were associated with a higher risk of mortality in sepsis patients. Measurement of CD4 iATP may serve as a useful tool for identifying patients at a higher risk of mortality and could potentially provide a basis for clinical treatment. Further research is warranted to fully elucidate the underlying mechanisms of this association.
Topics: Humans; Adenosine Triphosphate; Sepsis; Male; Female; CD4-Positive T-Lymphocytes; Middle Aged; Prospective Studies; Aged; Biomarkers; Prognosis; Intensive Care Units; Adult
PubMed: 38860755
DOI: 10.1002/iid3.1286 -
Journal of Cellular and Molecular... Jun 2024Ischemic stroke is one of the main causes of disability and death. However, recanalization of occluded cerebral arteries is effective only within a very narrow time...
Ischemic stroke is one of the main causes of disability and death. However, recanalization of occluded cerebral arteries is effective only within a very narrow time window. Therefore, it is particularly important to find neuroprotective biological targets for cerebral artery recanalization. Here, gene expression profiles of datasets GSE160500 and GSE97537 were downloaded from the GEO database, which were related to ischemic stroke in rats. Olfactory receptor 78 (Olfr78) was screened, and which highly associated with Calcium signalling pathway and MAPK pathway. Interacting protein of Olfr78, Prkaca, was predicted by STRING, and their interaction was validated by Co-IP analysis. Then, a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a neuronal cell model stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) were constructed, and the results showed that expression of Olfr78 and Prkaca was downregulated in MCAO rats and OGD/R-stimulated neurons. Overexpression of Olfr78 or Prkaca inhibited the secretion of inflammatory factors, Ca overload, and OGD/R-induced neuronal apoptosis. Moreover, Overexpression of Prkaca increased protein levels of cAMP, PKA and phosphorylated p38 in OGD/R-stimulated neurons, while SB203580, a p38 inhibitor, treatment inhibited activation of the cAMP/PKA-MAPK pathway and counteracted the effect of Olfr78 overexpression on improvement of neuronal functions. Meanwhile, overexpression of Olfr78 or Prkaca markedly inhibited neuronal apoptosis and improved brain injury in MCAO/R rats. In conclusion, overexpression of Olfr78 inhibited Ca overload and reduced neuronal apoptosis in MCAO/R rats by promoting Prkaca-mediated activation of the cAMP/PKA-MAPK pathway, thereby improving brain injury in cerebral ischaemia-reperfusion.
Topics: Animals; Reperfusion Injury; Rats; Male; Cyclic AMP; Rats, Sprague-Dawley; Receptors, Odorant; Apoptosis; Brain Ischemia; MAP Kinase Signaling System; Cyclic AMP-Dependent Protein Kinases; Brain Injuries; Neurons; Disease Models, Animal; Infarction, Middle Cerebral Artery; Signal Transduction
PubMed: 38856956
DOI: 10.1111/jcmm.18366 -
Clinical and Experimental Medicine Jun 2024Lung transplant (LTx) recipients face a significant risk from coronavirus disease 2019 (COVID-19), with elevated hospitalization mortality rates even post-vaccination....
Lung transplant (LTx) recipients face a significant risk from coronavirus disease 2019 (COVID-19), with elevated hospitalization mortality rates even post-vaccination. While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) typically induces pneumonia in even healthy individuals, it can also infect the transplanted lungs of LTx recipients, potentially leading to graft dysfunction. Despite the prevalence of COVID-19 pneumonia in LTx recipients, data on its characteristics and associated risk factors remain limited. This retrospective study analyzed data from LTx recipients at Tohoku University Hospital between January 2001 and November 2023. COVID-19 cases were identified, and patient records, including thoracic computed tomography (CT) evaluations, were reviewed. Patient characteristics, vaccination history, immunosuppressant use, and comorbidities were assessed. Descriptive analysis was utilized for data presentation. Among 172 LTx recipients, 39 (22.7%) contracted COVID-19, with 9 (23%) developing COVID-19 pneumonia. COVID-19 incidence in LTx recipients aligned with national rates, but pneumonia risk was elevated. Delayed antiviral therapy initiation was noted in pneumonia cases. Remdesivir was uniformly administered and remained the primary treatment choice. LTx recipients are susceptible to COVID-19 pneumonia, warranting vigilance and tailored management strategies. Pre-transplant vaccination and prompt COVID-19 diagnosis and treatment are imperative for optimizing outcomes in this population.
Topics: Humans; COVID-19; Male; Female; Japan; Middle Aged; Lung Transplantation; Risk Factors; Retrospective Studies; Aged; Adult; Antiviral Agents; SARS-CoV-2; Transplant Recipients; Treatment Outcome; COVID-19 Drug Treatment; Incidence; Adenosine Monophosphate; Alanine
PubMed: 38856777
DOI: 10.1007/s10238-024-01388-y -
The Journal of Cell Biology Sep 2024Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through...
Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.
Topics: Animals; Mice; Cilia; Cyclic AMP; Hedgehog Proteins; Mice, Knockout; Prostaglandins; Receptors, Prostaglandin E, EP4 Subtype; Signal Transduction
PubMed: 38856684
DOI: 10.1083/jcb.202306002 -
Brain Research Bulletin Aug 2024Limb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with...
BACKGROUND
Limb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with PF can achieve better therapeutic effect is unknown.
PURPOSE
This study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats.
METHODS
Middle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47 in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 μM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47 (ser 304 and ser 345) were detected.
RESULTS
LRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47 (ser 304 and ser 345) in neutrophils.
CONCLUSION
The protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.
Topics: Animals; Neutrophils; Male; Ischemic Postconditioning; Reperfusion Injury; Glucosides; Rats, Sprague-Dawley; Rats; Monoterpenes; Brain Ischemia; NADPH Oxidases; Infarction, Middle Cerebral Artery; p38 Mitogen-Activated Protein Kinases; NADP; Signal Transduction
PubMed: 38852654
DOI: 10.1016/j.brainresbull.2024.111006